Biomedical subjects

Ken Lukowiak

Publications and source records attributed to Ken Lukowiak.

At least 37 records · Page 2Linked to original sources

PubMed2004

Learning and memory in Lymnaea are negatively altered by acute low-level concentrations of hydrogen sulphide.

Hydrogen sulphide (H(2)S) is a common industrial pollutant as well as an endogenous neural transmitter/neural modulator. Experiments were performed on the pond snail Lymnaea stagnalis to determine the acute effects of low-level exposure to H(2)S (50-100 micromol l(-1)) on aerial respiratory behaviour, associative learning, and its subsequent consolidation into long-term memory (LTM). A 3-neuron network whose sufficiency and necessity have been demonstrated drives aerial respiratory behaviour in Lymnaea. In the presence of 100 micromol l(-1) H(2)S the number of bouts of aerial respiration and the total breathing time were significantly increased compared to the control hypoxic situation, but were equivalent to those observed in snails that had been subjected to a 'more intense hypoxic challenge'. In addition, at a concentration of 100 micromol l(-1) H(2)S neither associative learning nor long-term memory (LTM) were observed. However, snails subjected to a 'more intense hypoxic challenge' still had the capacity to learn and form LTM. These snails, in fact, showed statistically the best learning and memory performance of any group. While learning and memory were observed at 50 and 75 micromol l(-1) H(2)S, respectively, they were statistically poorer than the learning and memory exhibited by snails in the standard hypoxia condition. Hence the ability to learn and form memory was compromised by H(2)S. Thus an invertebrate model system with a well-defined neural network can be used to study of the effects of H(2)S on the processes of learning and memory.

Analysis of Variance↗

PubMed2003

Extinction requires new RNA and protein synthesis and the soma of the cell right pedal dorsal 1 in Lymnaea stagnalis.

Lymnaea stagnalis were operantly conditioned to not perform aerial respiratory behavior. This learned response was subsequently extinguished. Here, we show that spaced extinction training is more effective than massed extinction training, in addition to the occurrence of spontaneous recovery. We also find evidence of a critical period within the first hour after extinction training in which new RNA and protein synthesis must occur for a memory of extinction training to be established. The memory for extinction training can also be extended using cooling and by preventing aerial respiration from occurring after extinction training. In addition, we demonstrate that memory formation of extinction training requires the soma of the cell right pedal dorsal 1, a cell that we have previously shown to be necessary for long-term memory consolidation and reconsolidation. This finding implies that the events that lead to the formation of extinction memory occur in the same cell that is responsible for long-term memory of operant conditioning. All of these data are consistent with the hypothesis that, during extinction, a new associative memory is being formed and that this new memory covers up, but does not abolish, the "old" memory.

Animals↗

PubMed2003

Reconsolidation of a long-term memory in Lymnaea requires new protein and RNA synthesis and the soma of right pedal dorsal 1.

Reconsolidation of a long-term memory (LTM) in the snail Lymnaea stagnalis can be disrupted by cooling, an RNA synthesis blocker (actinomycin D), and by specifically ablating the soma of a cell we know is a site of LTM consolidation (right pedal dorsal 1, RPeD1). Aerial respiratory behavior was conditioned operantly by applying a gentle tactile stimulus to the pneumostome area (the respiratory orifice) every time the snail began to open its pneumostome to perform aerial respiration. This resulted in a reduction of this behavior while leaving cutaneous respiration intact. One week after training one-half of the animals received a memory reactivation session, which was similar to the original training (i.e., animals received reinforcement). All animals then received 1 hr of cooling, an injection of actinomycin D or saline, or the soma ablation procedure. This was followed by a test for savings 4 hr or 4 d later, which was also similar to the original training. Only those animals that received both the memory reactivation session and the treatment showed memory impairment during the test for savings. That is, the impairment was contingent on memory reactivation. These data indicate that reconsolidation requires both new RNA and protein synthesis to stabilize a reactivated memory, and it demonstrates that the soma of RPeD1, a cell that we have shown previously to be required in the consolidation of an LTM, is necessary for reconsolidation. These data suggest that the critical molecular processes occurring during both consolidation and reconsolidation transpire in the same cell in Lymnaea.

Animals↗

PubMed2003

Electrophysiological differences in the CpG aerial respiratory behavior between juvenile and adult Lymnaea.

Intact, freely moving juvenile Lymnaea perform aerial respiration significantly less often than do adults. We therefore hypothesized that RPeD1, the central pattern generator (CPG) neuron that initiates rhythmogenesis, would be less active in juveniles than adults. Using both isolated and semi-intact preparations to directly test this hypothesis, we found the opposite; juvenile RPeD1s were significantly smaller and more excitable than RPeD1s from adults. Significant age-related differences were found in the membrane resistance (greater in juveniles), time constant (smaller in juveniles), and rheobase current (lower in juveniles), all of which would tend to make juvenile cells significantly more excitable. However, there were significant age-related differences in the synaptic connectivity within the CPG and in peripheral input to the CPG, all which favor more rhythmic activity in the adult CPG. As was the case for intact Lymnaea, juvenile semi-intact preparations perform aerial respiration less often than do adults. The difference in excitability between juvenile and adult RPeD1s is therefore not sufficient to cause increased rhythmogenesis. Age-related changes in synaptic connectivity within the respiratory CPG and in peripheral modulation allow respiratory rhythmogenesis to be more easily expressed in adults which may compensate for their decreased dependence on cutaneous respiration as their surface to volume ratio changes as the grow in size.

Action Potentials↗

PubMed2003

Generation of functional inhibitory neurons in the adult rat hippocampus.

Several thousand new neurons are produced each day in the adult mammalian hippocampus, among which only excitatory granule cells (GCs) have thus far been identified. In the present study, we used mutant Semliki Forest Virus vectors to express enhanced green fluorescent protein in the hippocampus, and observed that approximately 14% of newly generated neurons in the dentate gyrus of adult rats are GABAergic basket cells (BCs). With the use of double whole-cell patch-clamp recordings from BC-GC pairs in hippocampal slices, we demonstrate that newly generated BCs in the dentate gyrus form inhibitory synapses with principal GCs. These data show for the first time that functional inhibitory neurons are recruited in the dentate gyrus of adult rats.

Animals↗

PubMed2003

Nitric oxide mediates metabolism as well as respiratory and cardiac responses to hypoxia in the snail lymnaea stagnalis.

Lymnaea stagnalis were exposed to hypoxic and chemical challenges while ventilation, heart rate and metabolism were monitored. Hypoxia increased ventilatory behavior, but this response was eliminated by immersion in 0.75 mM nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7 NI). 7 NI also suppressed ventilatory behavior under normoxia. 10.0 mM L-arginine (ARG, the NOS substrate) increased ventilatory behavior under normoxia, but dampened the hypoxic response. The heart-rate response to NOS inhibition exhibited dose-dependent contradictory characteristics. Under both normoxia and hypoxia 0.25 mM 7 NI increased heart rate, while 0.75 mM 7 NI suppressed it. The effect of 0.50 mM 7 NI depended on whether normoxia or hypoxia was coincident; under normoxia 0.50 mM 7 NI increased heart rate, while under hypoxia this concentration suppressed heart rate. Exposure to ARG did not elicit dose-dependent contradictory responses. Heart rate increased when treated with 10.0 mM ARG under normoxia and hypoxia, while 1.0 mM ARG increased heart rate only under hypoxia. Metabolic responses to NOS inhibition also exhibited dose-dependent contradictory changes. V.O2 decreased over 60% in response to 0.75 mM 7 NI, and baseline V.O2 was restored when exposure ceased. In contrast, 0.25 mM 7 NI increased V.O2 10%, and the increase continued after exposure ceased. 0.50 mM 7 NI decreased V.O2 40%, but V.O2 increased when exposure ceased. ARG had only the effect of increasing V.O2, and only at 10.0 mM concentration. Based on these results and on NO's known role as a neuromodulator, we conclude that the cardio-respiratory responses to hypoxia are, in part, mediated by NO.

Animals↗

PubMed2003

A molluscan model system in the search for the engram.

A 3-neuron central pattern generator, whose sufficiency and necessity has been directly demonstrated, mediates aerial respiratory behaviour in the pond snail, Lymnaea stagnalis. This behaviour can be operantly conditioned, and this associative learning is consolidated into long-lasting memory. Depending on the operant conditioning training procedure used the learning can be consolidated into intermediate term (ITM) or long-term memory (LTM). ITM persists for only 2-3 h, whilst LTM persists for days to weeks. LTM is dependent on both altered gene activity and new protein synthesis while ITM is only dependent on new protein synthesis. We have now directly established that one of the 3-CPG neurons, RPeD1, is a site of LTM formation and storage. We did this by ablating the soma of RPeD1 and leaving behind a functional primary neurite capable of mediating the necessary synaptic interactions to drive aerial respiratory behaviour by the 3-neuron CPG. However, following soma ablation the neuronal circuit is only capable of mediating learning and ITM. LTM can no longer be demonstrated. However, if RPeD1's soma is ablated after LTM consolidation memory is still present. Thus the soma is not needed for the retention of LTM. Using a similar strategy it may be possible to block forgetting.

Animals↗

PubMed2003

Cooling blocks ITM and LTM formation and preserves memory.

In Lymnaea aerial respiratory behaviour can be operantly conditioned; snails learn not to perform this behaviour. Depending on the training procedure used, snails are competent to form either intermediate-term (ITM; lasting 1-3 h) or long-term (LTM; >4 h) memory. We found that cooling the snails for 1 h immediately after training was sufficient to block either ITM or LTM. Cooling snails for a similar period 10 or 15 min after cessation of training, failed to block ITM and LTM formation, respectively. Finally, we employed the cooling technique to extend both ITM and LTM. That is, cooling could prevent forgetting. Cooling extended LTM that normally persisted for 2 days to at least 8 days. These data are consistent with the hypothesis that forgetting is due to the learning and remembering of interfering events, and thus is an active process.

Animals↗

PubMed2003

Forgetting and the extension of memory in Lymnaea.

Aerial respiratory behaviour in Lymnaea stagnalis was operantly conditioned using a procedure that results in long-term memory (LTM) persisting for 1 but not 3 days. By manipulating the snails' post-training environment, i.e. preventing Lymnaea from performing aerial respiratory behaviour, memory persistence was significantly extended. Memory retention, however, is only extended if snails are prevented from performing aerial respiration in the same context in which they were trained. Snails trained in the 'standard' context but prevented from performing aerial respiration in the 'carrot-odor' context (and vice versa) did not extend their memory. These data are consistent with the hypothesis that forgetting is due to interfering events, that occur following learning and memory consolidation.

Animals↗

PubMed2003

Intermediate and long-term memories of associative learning are differentially affected by transcription versus translation blockers in Lymnaea.

Aerial respiratory behaviour in the pond snail, Lymnaea stagnalis, can be operantly conditioned. This associative learning then undergoes consolidation into a long-lasting memory which, depending on the training procedure used, causes intermediate-term memory (ITM; lasting 3 h) or long-term memory (LTM; lasting >6 h) to be formed. We determined the differential susceptibility of these two forms of memory to translation and transcription blockers. The injection of a translation blocker, Anisomycin, 2.5 h before training prevents the establishment of both ITM and LTM. On the other hand, injection of the transcription blocker Actinomycin D, 2.5 h before training, did not prevent the establishment of ITM, but did, however, prevent LTM formation. Thus in Lymnaea, following associative learning, both ITM and LTM are dependent on new protein synthesis. ITM appears to be dependent on protein synthesis from preexisting transcription factors, whilst LTM is dependent on protein synthesis from new transcription messages.

Animals↗

PubMed2003

Associative learning and memory in Lymnaea stagnalis: how well do they remember?

The search for 'the how and the where' of memory formation in the brain, the engram, is still one of the unattained 'Holy Grails' of neuroscience. Over the years, various paths have been trodden in attempts to attain this goal, and while tantalizing glimpses appear now and then on the scientific horizon, the Grail still has not been grasped. One of the paths that investigators have walked is the invertebrate 'model system' approach. Some invertebrates possess relatively simple nervous systems that mediate relatively simple behaviours that are both interesting and trainable. In this commentary, we would like to shed light on a relatively new player, the pond snail Lymnaea stagnalis L., that is being used in the quest to illuminate 'the how and the where' the nervous systems encode and store memory. We will show that it is possible to demonstrate that a single neuron is a site of memory formation and storage for a form of associative learning in this lowly snail. It may be that the Grail is a little closer to being grasped.

Animals↗

PubMed2002

Development of Ca2+ hotspots between Lymnaea neurons during synaptogenesis.

Calcium (Ca2+) channel clustering at specific presynaptic sites is a hallmark of mature synapses. However, the spatial distribution patterns of Ca2+ channels at newly formed synapses have not yet been demonstrated. Similarly, it is unclear whether Ca2+ 'hotspots' often observed at the presynaptic sites are indeed target cell contact specific and represent a specialized mechanism by which Ca2+ channels are targeted to select synaptic sites. Utilizing both soma-soma paired (synapsed) and single neurons from the mollusk Lymnaea, we have tested the hypothesis that differential gradients of voltage-dependent Ca2+ signals develop in presynaptic neuron at its contact point with the postsynaptic neuron; and that these Ca2+ hotspots are target cell contact specific. Fura-2 imaging, or two-photon laser scanning microscopy of Calcium Green, was coupled with electrophysiological techniques to demonstrate that voltage-induced Ca2+ gradients (hotspots) develop in the presynaptic cell at its contact point with the postsynaptic neuron, but not in unpaired single cells. The incidence of Ca2+ hotspots coincided with the appearance of synaptic transmission between the paired cells, and these gradients were target cell contact specific. In contrast, the voltage-induced Ca2+ signal in unpaired neurons was uniformly distributed throughout the somata; a similar pattern of Ca2+ gradient was observed in the presynaptic neuron when it was soma-soma paired with a non-synaptic partner cell. Moreover, voltage clamp recording techniques, in conjunction with a fast, optical differential perfusion system, were used to demonstrate that the total whole-cell Ca2+ (or Ba2+) current density in single and paired cells was not significantly different. However, the amplitude of Ba2+ current was significantly higher in the presynaptic cell at its contact side with the postsynaptic neurons, compared with non-contacted regions. In summary, this study demonstrates that voltage-induced Ca2+ hotspots develop in the presynaptic cell, concomitant with the appearance of synaptic transmission between the soma-soma paired cells. The appearance of Ca2+ gradients in presynaptic neurons is target cell contact specific and is probably due to a spatial redistribution of existing channels during synaptogenesis.

Animals↗

PubMed2002

Specificity of synapse formation between Lymnaea heart motor neuron and muscle fiber is maintained in vitro in a soma-muscle configuration.

Precise neuronal connectivity during development is subservient to all nervous system functions in adult animals. However, the cellular mechanisms that mastermind this neuronal connectivity remain largely unknown. This lack of fundamental knowledge regarding nervous system development is due in part to the immense complexity of mammalian brain, as cell-cell interactions between defined sets of pre- and postsynaptic partners are often difficult to investigate directly. In this study, we developed a novel model system which has allowed us to reconstruct synapses between identified motor neurons and their target heart muscle cell in a soma-muscle configuration. Utilizing this soma-myocardial cell synapse model, we demonstrate that synapses between somata and heart muscle cells can be reconstructed in cell culture. The soma-myocardial cell synapses required 12-24 h to develop and thus differed temporally from conventional neuromuscular synapses (seconds to a few minutes). We also demonstrate that the synapses are target cell-type-specific and are most likely independent of transmitter phenotypic characteristics of presynaptic neurons.

Animals↗

PubMed2002

Context extinction and associative learning in Lymnaea.

Aerial respiratory behavior in the pond snail Lymnaea was operantly conditioned so that snails learned not to perform aerial respiration in a hypoxic environment. Snails were trained in either the standard context (no food odorant) or a carrot (food-odorant) context. An operant training procedure of two 45-min training sessions with a 1-h interval between the sessions followed by a third 45-min training session 18 h later was sufficient to produce associative learning and long-term memory (LTM) that persisted for at least 5 days. If, however, following the third operant training session snails received three 45-min extinction training sessions, with each extinction session separated by at least a 1-h interval, LTM was not observed when tested the following day. That is, the memory was extinguished. Extinction, however, did not occur if the context of the extinction training was different from the context of the associative training. That is, in the snails trained in the standard context, extinction did not occur if the extinction training sessions were performed in the food-odorant context and vice versa.

Animals↗

PubMed2002

The Soma of RPeD1 must be present for long-term memory formation of associative learning in Lymnaea.

The cellular basis of long-term memory (LTM) storage is not completely known. We have developed a preparation where we are able to specify that a single identified neuron, Right Pedal Dorsal 1 (RPeD1), is a site of LTM formation of associative learning in the pond snail, Lymnaea stagnalis. We demonstrated this by ablating the soma of the neuron but leaving behind its functional primary neurite, as evidenced by electrophysiological and behavioral analyses. The soma-less RPeD1 neurite continues to be a necessary participant in the mediation of aerial respiratory behavior, associative learning, and intermediate-term memory (ITM); however, LTM cannot be formed. However, if RPeD1's soma is ablated after LTM consolidation has occurred, LTM can still be accessed. Thus the soma of RPeD1 is a site of LTM formation.

Animals↗

PubMed2002

Changes in the activity of a CpG neuron after the reinforcement of an operantly conditioned behavior in Lymnaea.

We have previously shown that the aerial respiratory behavior of the mollusk Lymnaea stagnalis can be operantly conditioned, and the central pattern generating (CPG) neurons underlying this behavior have been identified. As neural correlates of operant conditioning remain poorly defined in both vertebrates and invertebrates, we have used the Lymnaea respiratory CPG to investigate neuronal changes associated with the change in behavior after conditioning. After operant conditioning of the intact animals, semi-intact preparations were dissected, so that changes in the respiratory behavior (pneumostome openings) and underlying activity of the identified CPG neuron, right pedal dorsal 1 (RPeD1), could be monitored simultaneously. RPeD1 was studied because it initiates the rhythmic activity of the CPG and receives chemo-sensory input from the pneumostome area. Pneumostome openings and RPeD1 activity were monitored both before and after a reinforcing training stimulus applied to the open pneumostome of operantly conditioned and yoked control preparations. After presentation of the reinforcing stimulus, there was a significant reduction in both breathing behavior and RPeD1 activity in operant preparations but not in yoked and naïve controls. Furthermore these changes were only significant in the subgroup of operantly conditioned animals described as good learners and not in poor learners. These data strongly suggest that changes in RPeD1 activity may underlie the behavioral changes associated with the reinforcement of operant conditioning of the respiratory behavior.

Animals↗

PubMed2002

Gone but not forgotten: the lingering effects of intermediate-term memory on the persistence of long-term memory.

Aerial respiratory behaviour can be operantly conditioned in Lymnaea stagnalis and, depending on the interval between the training sessions, memories of significantly different durations are produced. In naïve snails, a 15 min training procedure with a 30 min interval between three training sessions results in memory that persists for only 3 h (intermediate-term memory, ITM); whilst if the three 15 min training sessions are separated by a 1 h interval memory persists for 48 h (long-term memory, LTM). We found that if ITM training preceded LTM training, then LTM would persist for 24 h longer. This augmenting effect on LTM persistence could be demonstrated for up to 5 h following the last ITM training session, even though ITM was not observed at that time. However, if LTM training ensued 8 h after the last ITM training session, an augmented LTM did not occur. Extinguishing the memory produced by the ITM training procedure also prevented augmentation of LTM. That is, if an extinction procedure was given to the snails after the ITM training procedure, LTM did not persist longer than 48 h. Thus, at the behavioural level, ITM and LTM are interconnected.

Animals↗

PubMed2002

The effects of continuous versus partial reinforcement schedules on associative learning, memory and extinction in Lymnaea stagnalis.

A continuous schedule of reinforcement (CR) in an operant conditioning procedure results in the acquisition of associative learning and the formation of long-term memory. A 50 % partial reinforcement (PR) schedule does not result in learning. The sequence of PR-CR training has different and significant effects on memory retention and resistance to extinction. A CR/PR schedule results in a longer-lasting memory than a PR/CR schedule. Moreover, the memory produced by the CR/PR schedule is resistant to extinction training. In contrast, extinction occurs following the PR/CR schedule.

Animals↗